Following obtaining support from the Director, NCI, Dr. Waldmann directed an inter-institute collaboration (NCI-NIAID) directed toward the production under current good manufacturing practices (cGMP) of IL-15 for clinical trials in patients with metastatic malignant melanoma and renal cell cancer as well as in patients with HIV/AIDS receiving HAART therapy. In a modification of an expression system and purification method developed in the Waldmann Laboratory, the Biopharmaceutical Development Program (BDP) of the National Cancer Institute under the direction of Steven Creekmore has produced recombinant human IL-15 (rhIL-15) under current good manufacturing practices (cGMP). In this work, an E. coli based fermentation and purification process was developed for the production of clinical grade recombinant human IL-15. DNA sequences from human IL-15 were inserted into pET-28b plasmid, were expressed in the E. coli host BL21-AL. The inclusion bodies of IL-15 produced in E. coli were solubilized, refolded and orthogonally purified to yield active IL-15. RhIL-15 was produced as a non-glyclosylated, single-chain polypeptide of 115 amino acids, having a calculated molecular weight of 12,901 Da. Purified rhIL-15 showed in vitro activity in a CTLL-2 cell proliferation assay in the range of 15-25 x 106 IU/mg protein standardized against the international standard obtained from the National Institute of Biological Standard and Control (NIBSC). In a toxicology study directed by the Waldmann Laboratory, the rhIL-15 was evaluated for safety (toxicity), pharmacokinetics, immunogenicity, autoimmunity and impact on elements of the normal immune system of rhIL-15 in rhesus macaques. The only biologically meaninful laboratory abnormally was a grade 3/4 neutropenia that was secondary to a redistribution of neutrophils from the circulation to the tissues. No abnormalities were observed following IL-15 administration in gross or microscopic analysis of tissues at autopsy. The animals did not produce antibodies to IL-15 as assessed using a sensitive 2-armed capture technique developed in the Waldmann Laboratory. IL-15 administration was associated with four to eight fold increases in the number of circulating NK and CD3 CD8 especially central and effector memory T-lymphocytes. The administration was also associated with a tenfold increase in the circulating IL-2R alpha levels. CDER FDA and IRB NCI approval has been granted to initiate a clinical trial Phase I Study of Intravenous Recombinant Human IL-15 (rhIL-15) in Adults with Refractory Metastatic Malignant Melanoma and Metastatic Renal Cell Cancer. The sponsor is the Metabolism Branch, CCR, NCI with Dr. Thomas A. Waldmann as Study Chair and IND holder. The study is being performed on the Metabolism Branch, CCR, NCI in the Clinical Center with Dr. Thomas Waldmann as the principal investigator (PI) and Kevin Conlon as the Principal Associate Investigator. The primary objective of this trial is to determine the safety, toxicity profile, dose-limiting toxicity and maximum tolerated dose of intravenous recombinant human IL-15 produced by the BDP and administered as a daily intravenous infusion for 12 days in individuals with incurable metastatic malignant melanoma and renal cell cancer. The secondary objectives include determination of rhIL-15 pharmacokinetics and to determine the immunogenicity of the IL-15 in individuals receiving this drug. Phenotypic analysis of circulating lymphocytes will be used to define the biological effects of rhIL-15. A final objective is to obtain preliminary information on the efficacy of IL-15 in the treatment of patients with metastatic malignancy. The first patient entered the trial by signing the consent form on 10/21/2010. This patient started treatment on 10/25/2010. Five patients have completed their course of therapy at 3.0 and three patients have been entered at the 1.0 mcg/kg/day dose. Following the initial as well as the 12th (last infusion) of IL-15 the serum concentration of IL-15 at 10 minutes following the infusion ranged from 20,000 to 90,000 picograms/mL. The level declined with an alpha phase of approximately 30 minutes for the first two hours and then declined with a beta phase with a survival T of about 2-3 hours. For the first 2-3 hours the very high levels of IL-15 were sufficient to signal though the IL-2/IL-15R beta and common gamma chain alone receptor systems shared with IL-2 (affinity 10-9). At 24 hours IL-15 was still detectable in the serum at levels of 25 to 100 picograms/mL, levels that would require the heterotrimeric receptor with its affinity of 10-11. The pharmacokinetics are clearly not optimal, therefore it would be of value to evaluate subcutaneous and continuous infusion dosing schedules especially in association with agonistic antibodies to CD40 to induce IL-15R alpha expression. Initially the patients were not premedicated to gain an insight into the timing of symptoms related to immune activation. There was a pattern of fever beginning 2 to 2 hours following the start of rhIL-15 infusions, peaking reliably at the 3 -hour time point. Treatment with acetaminophen 650 mg to 1,000 mg every 6 hours with 2 doses of ibuprofen 500 mg at one and seven-hour time points became standard medication. Rigors occurred reliably at the 4-hour time points. This was treated with IV Meperidine 25 to 75 mg. Rigors and fever were also observed at the 1.0 mcg/kg/day dose but were less severe. The patients treated at 3.0 mcg/kg/day but not those treated at 1.0 mcg/kg/day established a pattern of transient hypotension in the period from 5 to 10 hours following the IL-15 infusion. This hypotension was controlled with i.v. normal saline. The total lymphocyte count routinely fell from its normal 1,000 to 2,000 mm3 to 200 to 600 mm3 on days two and three and then rose in the period from days eight through twelve to approximately 4,000 mm3. The NK cell numbers during the pretherapy-evaluation period ranged from the absolute numbers of 200 to 400 mm3. By day eight the absolute NK numbers rose to 1,000-1,100 and then increased further so that by day 14 their levels were 2,600 mm3, an increase of over eightfold from the pretherapy levels. Examination of the blood smear revealed activated granules containing natural killer (NK) cells. They were also increases in the numbers of CD122 expressing cells and in the number of CD8+ CD45RO CCR7- effector memory T cells. There was a 22% reduction in the size of the malignant melanoma lesions. Many of the patients in the trial had received high-dose IL-2 and TIL therapy and had failed these approaches, thus they are unlikely to respond to IL-15. In addition there was a reduction in the size of renal cell cancer lung metastases that continued to decline in size following cessation of treatment. We wish to complete the phase I trial with bolusIL-15 injections and then evaluate both daily subcutaneous (5x/week/x 2 weeks) on continuous I.V. (for 10 days) dosing strategies.